We would like to thank the Wellcome Trust Sanger Institute for the gifts of piggyBac transposon and piggyBac transposase. This work was supported by the Butcher grant of Colorado and the NIH R01 AG041801 (W.S.C).

<ol>
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The authors have nothing to disclose.

Forward genetic analysis allows for an unbiased interrogation of the genome for genes responsible for a specific phenotype. This method is very powerful to uncover novel gene functions. It has been widely used in lower organisms but not in mammal, such as the mouse, mainly due to the extremely high cost associated with the infrastructure and logistics that would entail. Here, we moved the genetic screening process to the ES cell culture platform, greatly increasing the efficiency and throughput in generating mutants a...

Ömür stres direnci ile samimi bir ilişki vardır. Genel olarak, uzun ömürlü türler genellikle hidrojen peroksit paraquatın (PQ), UV, ısı ve ağır metaller 1,2 gibi birden stres, doğru direncin artmasına işlemini göstermektedir. Bunun aksine, strese karşı artan hassasiyet, kısalmış ömür ve / veya daha fazla hastalık eğilimli fenotipi tahmin etmek eğilimindedir. Anti-oksidanın, tutucu yolu uzun hayvana gerilme direnci veren önemli bir rol oynadığı tahmin edilmektedir. Bununla birlikte, birkaç istisna dışında, çeşitli anti-oksidan enzimler (örneğin, akciğer), manipülasyonlara sahip transgenik hayvanların çeşitli çalışmalar, oksitleyici kirleticilerin alınmasına yönelik enzim düzeyi artan yaşam süresi ya da sağlık aralığı 3 arttırmadığını gösterir. Bu veriler sürekli olarak uzun ömürlü hayvanlarda gözlenen stres direnç özelliği henüz ortaya için diğer hücresel yolların aracılık ettiğini göstermektedir. Biz tarafsız bir ileriye aldıGenetik mutasyona uğramış bir yaklaşım üzerine, kültürlü embriyonik kök (ES) hücrelerde stres direnci fenotipi olabilir genleri tanımlamak için. ES hücreleri Bu çalışmada iki ana avantaj sağlar: (1) sofistike genetik manipülasyonlar ES hücrelerinin genomunun modifiye edilmesi için kullanılabilir; ve (2) ekranında elde herhangi bir strese karşı ES hücreleri ile doğrudan ömrü, sağlık yayılma ölçmek için bütün hayvan çalışmalarında içine hızlı şekilde çevrilmesine izin veren fare üretimi için de kullanılabilir. Bu yazıda Blm alleller bir tetrasiklin duyarlı eleman tarafından kontrol altına bulundukları C9 ES hücre hattı kullanımını tarif. Doksisiklin (Ortodoks) tedavisi geçici kardeş kromatid değişimi artan olaya lider BLM ifadesini kapalı. Bu kısa vadeli Blm knock-out heterozigot nüfus içerisindeki homozigot mutasyon üretimi için izin stres direnci için çekinik mutasyonlar tarama sürecinde yakalanan olabilir ki. BizAyrıca rastgele genom genlerin mutasyona bir poli-A tuzak kaseti (PB-UPA) eklemek için mutajen olarak piggyBac (PB) transpozonun kullanımını tarif. Poli-A tuzağı ile bir gen bozulması olan hücreler, G418 dirençli hale ve gen-trap mutantlar (gen trap kütüphanesi) oluşan bir koleksiyon yapılmış ve daha sonra, karşı stres olan mutant klonları için tarandı olabilir, böylece elde edilebilir. Seçim kurtarıldı Stres dirençli klonlar eklemeleri sayısı (qPCR) arasında bağlamda moleküler tekniklerle oldukça hızlı karakterize edilebilir, ekleme site (splinkerette PCR), bozulmuş gen (ŞOK) kimliği ve onun ifade seviyesi ( RT-qPCR). PB yerleştirme Yabani tür bir DNA dizisini ve bu nedenle geri stres direnci kaybını test klonu MPB transpozazın geçici ifade yolu ile remobilized edilebilir. Bunlar önce yapılmalıdır mutasyon nedenselliğini onaylamak için güçlü yollar vardırpahalı bir fare üretimi. Önceki çalışmalar stres maruz kalan hücreler pluripotency 4,5 kaybettiklerini gösterdi. Böylece, bu protokolde, stres ile tedavi olmaz mutant hücreler kümesi bir yineleme, korunması başarılı fare üretimi için kritik öneme sahiptir. Bizim laboratuvar hem istek üzerine diğer araştırmacılar için kullanılabilir, C9 ES hücre hattı ve PB-UPA vektörü tasarladı. Burada rapor edilen protokol, stres dayanıklı klonların (Şekil 1B) izole etmek için replika plaka ve stres seçimi, ardından PB-UPA (Şekil 1A) gen tuzak ES hücreleri, de novo arşivinin oluşturulması yoluyla başlar. Biz paraquat, hücreler içinde güçlü bir serbest kök üretici madde ile noktası göstermiştir. Hemen hemen, örneğin herhangi bir sitotoksik bileşik veya toksin, ER stres (örneğin, thapsigargin ve tunikamisin), nöronal oksitleyici (örneğin, MPP +, 6-hidroksi dopamin ve rotenon), ısı ve ağırmetaller (örneğin, Cd, Se), ilgili dirençli mutantların seçmek için bir yöntem adapte edilebilir.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Vector&lt;/strong&gt;</td></tr><tr><td>PB-UPA</td><td></td><td></td><td></td></tr><tr><td>mPBase</td><td></td><td></td><td></td></tr><tr><td>mPBase&lt;sup&gt;Puro&lt;/sup&gt;</td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Tissue Culture&lt;/strong&gt;</td></tr><tr><td>500-ml Stericup filters</td><td>EMD Millipore</td><td>SCGPU05RE</td><td></td></tr><tr><td>250-ml Stericup filters</td><td>EMD Millipore</td><td>SCGPU02RE</td><td></td></tr><tr><td>50-ml Steriflip-GV filters</td><td>EMD Millipore</td><td>SE1M179M6</td><td></td></tr><tr><td>KO DMEM</td><td>Life Technologies</td><td>10829-018</td><td></td></tr><tr><td>DMEM</td><td>Sigma-Aldrich</td><td>D6429</td><td></td></tr><tr><td>FBS</td><td>Tissue Culture Biologicals</td><td>104</td><td></td></tr><tr><td>Heat Inactivated FBS</td><td>Sigma-Aldrich</td><td>F4135-500</td><td></td></tr><tr><td>LIF</td><td>EMD Millipore</td><td>ESG1107</td><td></td></tr><tr><td>Non-essential Amino Acids</td><td>Life Technologies</td><td>11140-050</td><td></td></tr><tr><td>GlutaMAX</td><td>Life Technologies</td><td>35050-061</td><td></td></tr><tr><td>Pen/Strep</td><td>Life Technologies</td><td>15140148</td><td></td></tr><tr><td>&amp;beta;-Mercaptoethanol</td><td>Life Technologies</td><td>21985-023</td><td></td></tr><tr><td>Methyl Viologen dichloride (Paraquat)</td><td>Sigma-Aldrich</td><td>856177</td><td></td></tr><tr><td>Dimethyl Sulphoxide Hybri-MAX</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>EmbryoMAX 0.1% gelatin</td><td>EMD Millipore</td><td>ES006B</td><td></td></tr><tr><td>DPBS/Modified</td><td>HyClone</td><td>SH30028.02</td><td></td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Life Technologies</td><td>25200-056</td><td></td></tr><tr><td>T25 Flask</td><td>Corning</td><td>353108</td><td></td></tr><tr><td>T75 flask</td><td>Corning</td><td>353135</td><td></td></tr><tr><td>100-mm&amp;nbsp; plate</td><td>Corning</td><td>353003</td><td></td></tr><tr><td>150-mm&amp;nbsp; plate</td><td>Corning</td><td>430599</td><td></td></tr><tr><td>96-well&amp;nbsp; plate</td><td>Corning</td><td>3585</td><td></td></tr><tr><td>96-well U-bottom plate</td><td>Corning</td><td>3799</td><td></td></tr><tr><td>24-well plate</td><td>Corning</td><td>3526</td><td></td></tr><tr><td>50-ml reservoir&amp;nbsp;</td><td>Corning</td><td>4870</td><td></td></tr><tr><td>15-ml tubes</td><td>VWR International, LLC</td><td>82050-276</td><td></td></tr><tr><td>Primary Mouse Embryonic Fibroblasts</td><td>EMD Millipore</td><td>PMEF-NL</td><td></td></tr><tr><td>DR4 Mouse Embryonic Fibroblasts</td><td>Applied StemCell</td><td>ASF-1001</td><td></td></tr><tr><td>Mitomycin C</td><td>Fisher BioReagents&amp;nbsp;</td><td>BP25312</td><td></td></tr><tr><td>Geneticin (G418)</td><td>Life Technologies</td><td>11811-023</td><td></td></tr><tr><td>Doxycycline</td><td>Fisher BioReagents&amp;nbsp;</td><td>BP26531</td><td></td></tr><tr><td>Cryotubes</td><td>Thermo Scientific</td><td>377267</td><td></td></tr><tr><td>Centrifuge&amp;nbsp;</td><td>Eppendorf</td><td>Centrifuge 5702</td><td></td></tr><tr><td>TC10 cell counter</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Counting Slides (for TC10)</td><td>Bio-Rad</td><td>1450011</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Electroporation&lt;/strong&gt;</td></tr><tr><td>Gene Pulser Xcell</td><td>Bio-Rad</td><td>1652611</td><td></td></tr><tr><td>Gene Pulser Cuvettes (4&amp;#160;mm gap)</td><td>Bio-Rad</td><td>1652088</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Molecular Biology&lt;/strong&gt;</td></tr><tr><td>Thermal Cycler</td><td>Eppendorf</td><td>Mastercylcer ep Gradient&amp;nbsp; S</td><td></td></tr><tr><td>Puregene Core kit B</td><td>Qiagen</td><td>158745</td><td></td></tr><tr><td>Topo-TA Cloning kit</td><td>Life Technologies&amp;nbsp;</td><td>450030</td><td></td></tr><tr><td>High Capacity cDNA synthesis kit</td><td>Applied Biosystems</td><td>4368814</td><td></td></tr><tr><td>NaCl</td><td>Fisher BioReagents&amp;nbsp;</td><td>BP358-212</td><td></td></tr><tr><td>100% ethanol</td><td>Decon Laboratories, Inc.</td><td>2716</td><td></td></tr><tr><td>Double Processed Tissue Culture Water</td><td>Sigma-Aldrich</td><td>W3500</td><td></td></tr><tr><td>Sau3A1</td><td>New England BioLabs</td><td>R0169L</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>New England BioLabs</td><td>M0202T</td><td></td></tr><tr><td>EcoRV</td><td>New England BioLabs</td><td>R3195S</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;96-well Lysis Buffer (Ramires-Solis &lt;em&gt;et al.&lt;/em&gt; 1992)&lt;/strong&gt;</td></tr><tr><td>Trizma Base</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Fisher BioReagents&amp;nbsp;</td><td>A144-212</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E5134</td><td></td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-Lauroylsarcosine sodium salt</td><td>Sigma-Aldrich</td><td>L5777</td><td></td></tr><tr><td>Proteinase-K</td><td>Fisher BioReagents&amp;nbsp;</td><td>BP1700</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Electrophoresis&lt;/strong&gt;</td></tr><tr><td>Mini-Sub Cell GT</td><td>Bio-Rad</td><td>170-4469EDU</td><td></td></tr><tr><td>LE Agarose&amp;nbsp;</td><td>GeneMate</td><td>E3120500</td><td></td></tr><tr><td>Ethidium Bromide&amp;nbsp;</td><td>Fisher BioReagents&amp;nbsp;</td><td>BP1302</td><td></td></tr><tr><td>100 BP DNA Ladder</td><td>New England BioLabs</td><td>N3231S</td><td></td></tr><tr><td>1 Kb DNA Ladder</td><td>New England BioLabs</td><td>N3232S</td><td></td></tr><tr><td>2-log DNA Ladder</td><td>New England BioLabs</td><td>N3200L</td><td></td></tr></tbody></table>

forward genetic approach to uncover stress resistance genes in mice &#8212; a high-throughput screen in es cells

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely limits its potential. We describe here the use of mouse embryonic stem (ES) cells as surrogate cells to screen for a phenotype of interest and subsequently introduce these cells into a host embryo to develop into a living mouse carrying the phenotype. This method provides (1) a cost effective, high-throughput platform for genetic screen in mammalian cells; (2) a rapid way to identify the mutated genes and verify causality; and (3) a short-cut to develop mouse mutants directly from these selected ES cells for whole animal studies. We demonstrated the use of paraquat (PQ) to select resistant mutants and identify mutations that confer oxidative stress resistance. Other stressors or cytotoxic compounds may also be used to screen for resistant mutants to uncover novel genetic determinants of a variety of cellular stress resistance.

Tipik bir mutajenez deneyde, transfeksiyon PB-UPA ve MPB transpozaz ile 3 x 10 7 ES hücrelerinin toplam oluşur. Iki bağımsız deneyin üretilen gen tuzak ES hücre sayısı, Tablo 1 &#39;de özetlenmiştir. Gen tuzak etkinliği, yaklaşık 0.04%&#39; dir. Kombine gen tuzak kütüphaneleri 22400 bağımsız mutantlar, fare genomu hakkında tam bir kapsama (23.000 kodlayan genlerin) içerirler. Aşırı kapsama tekrarlanan mutasyon yoluyla daha mutantlar üreten ve / veya süreci ölçeklem...

Watch this Scientific Journal Video about Forward Genetic Approach to Uncover Stress Resistance Genes in Mice Ã¢â‚¬â€ A High-throughput Screen in ES Cells at JoVE.com

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &#8212; A High-throughput Screen in ES Cells

Stres direnci uzun ömürlü özelliklerinden biridir ve genetik yönetilmeye bilinmektedir. Burada, uzun ömürlü çalışmaları için fare modelleri geliştirmek için hangi ES hücreleri stres direnci kazandıran mutasyonlar taranması için tarafsız bir yüksek verimli bir yöntem geliştirdi.

İleri Genetik Yaklaşım Farelerde Stres Direnci Genler ortaya çıkarmak için - ES Hücrelerde bir Yüksek verim Ekranı

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely ...

forward-genetic-approach-to-uncover-stress-resistance-genes-mice-high

Research

JoVE Journal

Developmental Biology

8.1K Görüntüleme.  University of Colorado Denver.  Stres direnci uzun ömürlü özelliklerinden biridir ve genetik yönetilmeye bilinmektedir. Burada, uzun ömürlü çalışmaları için fare modelleri geliştirmek için hangi ES hücreleri stres direnci kazandıran mutasyonlar taranması için tarafsız bir yüksek verimli bir yöntem geliştirdi. 

Video: Forward Genetic Approach to Uncover Stress Resistance Genes in Mice — A High-throughput Screen in ES Cells

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome editing, embryonic stem (ES) cell-based gene-targeting technology does not have these shortcomings and is widely used to modify animal/mouse genome ranging from large deletions/insertions to single nucleotide substitutions. Notably, identifying the relatively few homologous recombination (HR) events necessary to obtain desired ES clones is a key step, which demands accurate and reliable methods. Southern blotting and/or conventional PCR are often utilized for this purpose. Here, we describe the detailed procedures of using those two methods to identify HR events that occurred in mouse ES cells in which the endogenous Myh9 gene is intended to be disrupted and replaced by cDNAs encoding other nonmuscle myosin heavy chain IIs (NMHC IIs). The whole procedure of Southern blotting includes the construction of targeting vector(s), electroporation, drug selection, the expansion and storage of ES cells/clones, the preparation, digestion, and blotting of genomic DNA (gDNA), the hybridization and washing of probe(s), and a final step of autoradiography on the X-ray films. PCR can be performed directly with prepared and diluted gDNA. To obtain ideal results, the probes and restriction enzyme (RE) cutting sites for Southern blotting and the primers for PCR should be carefully planned. Though the execution of Southern blotting is time-consuming and labor-intensive and PCR results have false positives, the correct identification by Southern blotting and the rapid screening by PCR allow the sole or combined application of these methods described in this paper to be widely used and consulted by most labs in the identification of genotypes of ES cells and genetically modified animals.

Here, we present a detailed protocol for identifying homologous recombination events that occurred in mouse embryonic stem cells using Southern blotting and/or PCR. This method is exemplified by the generation of nonmuscle myosin II genetic replacement mouse models using traditional embryonic stem cell-based homologous recombination-mediated targeting technology.

Fare embriyonik kök hücre kullanarak Southern blot ve polimeraz zincir reaksiyonu Homolog rekombinasyon olaylar tanımlaması

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome ...

Genetik

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.

Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein.

Recombinase aracılı Kaset Değişimi Kullanarak Fare Embriyonik Kök Hücre yapı-fonksiyon Çalışmaları

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

Gelişim Biyolojisi

Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. The CRISPR/Cas9 system allows researchers to modify the genome with unprecedented efficiency, fidelity, and simplicity. Harnessing this technology, researchers are seeking a rapid, efficient, and easy protocol for generating GM mice. Here we introduce an improved method for cryopreservation of one-cell embryos that leads to a higher developmental rate of the freeze-thawed embryos. By combining it with optimized electroporation conditions, this protocol allows for the generation of knockout and knock-in mice with high efficiency and low mosaic rates within a short time. Furthermore, we show a step-by-step explanation of our optimized protocol, covering CRISPR reagent preparation, in vitro fertilization, cryopreservation and thawing of one-cell embryos, electroporation of CRISPR reagents, mouse generation, and genotyping of the founders. Using this protocol, researchers should be able to prepare GM mice with unparalleled ease, speed, and efficiency.

Here, we present a modified method for cryopreservation of one-cell embryos as well as a protocol that couples the use of freeze-thawed embryos and electroporation for the efficient generation of genetically modified mice.

Genetiği Değiştirilmiş Farelerin Yüksek Verimli Üretimi için Dondurulmuş Çözülmüş Embriyoların Kullanımı

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. ...

Application of Mouse Parthenogenetic Haploid Embryonic Stem Cells as a Substitute of Sperm

In organisms with sexual reproduction, germ cells are the source of totipotent cells that develop into new individuals. In mice, fertilization of an oocyte by a spermatozoon creates a totipotent zygote. Recently, several publications have reported that haploid embryonic stem cells (haESCs) can be a substitute for gametic genomes and contribute to embryos, which develop into mice. Here, we present a protocol to apply parthenogenetic haESCs as a substitute of sperm to construct embryos by intracytoplasmic injection into oocytes. This protocol consists of steps for preparing haESCs as sperm replacement, for injection of haESC chromosomes into oocytes, and for culture of semi-cloned embryos. The embryos can yield fertile semi-cloned mice after embryo transfer. Using haESCs as sperm replacement facilitates genome editing in the germline, studies of embryonic development, and investigation of genomic imprinting.

This article aims to demonstrate the use of parthenogenetic haploid embryonic stem cells as a substitute for sperm for the construction of semi-cloned embryos.

Sperm Yerine Fare Partenogenetik Haploid Embriyonik Kök Hücrelerin Uygulanması

In organisms with sexual reproduction, germ cells are the source of totipotent cells that develop into new individuals. In mice, fertilization of an ...

CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the efficiency in developing gene-knockout mice by inducing small indel mutation would be sufficient enough, the efficiency of embryo genome editing for making large-size DNA knock-in (KI) is still low. Therefore, in contrast to the direct KI method in embryos, gene targeting using embryonic stem cells (ESCs) followed by embryo injection to develop chimera mice still has several advantages (e.g., high throughput targeting in vitro, multi-allele manipulation, and Cre and flox gene manipulation can be carried out in a short period). In addition, strains with difficult-to-handle embryos in vitro, such as BALB/c, can also be used for ESC targeting. This protocol describes the optimized method for large-size DNA (several kb) KI in ESCs by applying CRISPR/Cas9-mediated genome editing followed by chimera mice production to develop gene-manipulated mouse models.

Here we present a protocol for developing genetically modified mouse models using embryonic stem cells, especially for large DNA knock-in (KI). This protocol is tuned up using CRISPR/Cas9 genome editing, resulting in significantly improved KI efficiency compared with the conventional homologous recombination-mediated linearized DNA targeting method.

Genle Manipüle Edilmiş Fare Modelleri Geliştirmek için Embriyonik Kök Hücrelerde CRISPR / Cas9 Aracılı Yüksek Verimli Gen Hedefleme

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the ...

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will greatly facilitate the use of ES cells for developmental biology studies, disease modeling, drug discovery, and regenerative medicine (reviewed in 1,2).
To expedite the identification and characterization of novel regulators of ES cell maintenance and self-renewal, we developed a fluorescence reporter-based assay to quantitatively measure the self-renewal status in mouse ES cells using the Oct4GiP cells 3. The Oct4GiP cells express the green fluorescent protein (GFP) under the control of the Oct4 gene promoter region 4,5. Oct4 is required for ES cell self-renewal, and is highly expressed in ES cells and quickly down-regulated during differentiation 6,7. As a result, GFP expression and fluorescence in the reporter cells correlates faithfully with the ES cell identity 5, and fluorescence-activated cell sorting (FACS) analysis can be used to closely monitor the self-renewal status of the cells at the single cell level 3,8.
Coupled with RNAi, the Oct4GiP reporter assay can be used to quickly identify and study regulators of ES cell maintenance and self-renewal 3,8. Compared to other methods for assaying self-renewal, it is more convenient, sensitive, quantitative, and of lower cost. It can be carried out in 96- or 384-well plates for large-scale studies such as high-throughput screens or genetic epistasis analysis. Finally, by using other lineage-specific reporter ES cell lines, the assay we describe here can also be modified to study fate specification during ES cell differentiation.

We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.

Fare Embriyonik Kök Hücre Bakım ve Kendini yenileme Regulate Genler Okumak Oct4GiP Reporter Assay

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will ...

Biyoloji

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrPSc) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that &gt;1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.

S. cerevisiae Protein bazlı miras için yüksek üretilen iş tarama

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived ...

Measurements of Physiological Stress Responses in C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPRER), the unfolded protein response of the mitochondria (UPRMT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Measurements of Physiological Stress Responses in C. Elegans

Here, we characterize cellular proteotoxic stress responses in the nematode C. elegans by measuring the activation of fluorescent transcriptional reporters and assaying sensitivity to physiological stress.

C. Eleganlarda Fizyolojik Stres Tepkilerinin Ölçüleri

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and ...

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

Identification of genetic interactions is a powerful tool to decipher the functions of gene(s) by providing insights into their functional relationships with other genes and organization into biological pathways and processes. Although the majority of the genetic screens were initially developed in Saccharomyces cerevisiae, a complementary platform for carrying out these genetic screens has been provided by Schizosaccharomyces pombe. One of the common approaches used to identify genetic interactions is by overexpression of clones from a genome-wide, high-copy-number plasmid library in a loss-of-function mutant, followed by selection of clones that suppress the mutant phenotype.
This paper describes a protocol for carrying out this &#39;multicopy suppression&#39;-based genetic screen in S. pombe. This screen has helped identify multicopy suppressor(s) of the genotoxic stress-sensitive phenotype associated with the absence of the Ell1 transcription elongation factor in S. pombe. The screen was initiated by transformation of the query ell1 null mutant strain with a high-copy-number S. pombe cDNA plasmid library and selecting the suppressors on EMM2 plates containing 4-nitroquinoline 1-oxide (4-NQO), a genotoxic stress-inducing compound. Subsequently, plasmid was isolated from two shortlisted suppressor colonies and digested by restriction enzymes to release the insert DNA. Plasmids releasing an insert DNA fragment were retransformed into the ell1 deletion strain to confirm the ability of these suppressor plasmid clones to restore growth of the ell1 deletion mutant in the presence of 4-NQO and other genotoxic compounds. Those plasmids showing a rescue of the deletion phenotype were sequenced to identify the gene(s) responsible for suppression of the ell1 deletion-associated genotoxic stress-sensitive phenotype.

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

This work describes a protocol for a multicopy suppressor genetic screen in Schizosaccharomyces pombe. This screen uses a genome-wide plasmid library to identify suppressor clone(s) of a loss-of-function phenotype associated with a query mutant strain. Novel genetic suppressors of the ell1 null mutant were identified using this screen.

Şizosakkaromices pombe'de Multicopy Suppressorların Tanımlanması için Genetik Ekran

Identification of genetic interactions is a powerful tool to decipher the functions of gene(s) by providing insights into their functional relationships ...

Deciphering High-Resolution 3D Chromatin Organization via Capture Hi-C

The spatial organization of the genome contributes to its function and regulation in many contexts, including transcription, replication, recombination, and repair. Understanding the exact causality between genome topology and function is therefore crucial and increasingly the subject of intensive research. Chromosome conformation capture technologies (3C) allow inferring the 3D structure of chromatin by measuring the frequency of interactions between any region of the genome. Here we describe a fast and simple protocol to perform Capture Hi-C, a 3C-based target enrichment method that characterizes the allele-specific 3D organization of megabased-sized genomic targets at high-resolution. In Capture Hi-C, target regions are captured by an array of biotinylated probes before downstream high-throughput sequencing. Thus, higher resolution and allele-specificity are achieved while improving the time-effectiveness and affordability of the technology. To demonstrate its strengths, the Capture Hi-C protocol was applied to the mouse X-inactivation center (Xic), the master regulatory locus of X-chromosome inactivation (XCI).

Deciphering High-Resolution 3D Chromatin Organization via Capture Hi-C

This protocol describes the Capture Hi-C method used to characterize the 3D organization of megabased-sized targeted genomic regions at high-resolution, including boundaries of topologically associating domains (TADs) and long-range chromatin interactions between regulatory and other DNA sequence elements.

Yüksek Çözünürlüklü 3D Kromatin Organizasyonunun Capture Hi-C ile Deşifre Edilmesi

The spatial organization of the genome contributes to its function and regulation in many contexts, including transcription, replication, recombination, ...